Chromatin loop domain organization within the 4q35 locus in facioscapulohumeral dystrophy patients versus normal human myoblasts

Chromatin loop domain organization within the 4q35locus in facioscapulohumeral dystrophy patientsversus normal human myoblastsAndrei Petrov*, Iryna Pirozhkova*, Gilles Carnac†, Dalila Laoudj†, Marc Lipinski*, and Yegor S. Vassetzky*‡

*Interactions Moleculaires et Cancer, Unite Mixte de Recherche 8126, Centre National de la Recherche Scientifique–Universite Paris-Sud 11–InstitutGustave-Roussy, F-94805 Villejuif, France; and †Centre de Recherches de Biochimie Macromoleculaire, 34293 Montpellier, France

Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved March 14, 2006 (received for review January 6, 2006)

Fascioscapulohumeral muscular dystrophy (FSHD) is an autosomaldominant neuromuscular disorder linked to partial deletion of inte-gral numbers of a 3.3 kb polymorphic repeat, D4Z4, within thesubtelomeric region of chromosome 4q. Although the relationshipbetween deletions of D4Z4 and FSHD is well established, how thistriggers the disease remains unclear. We have mapped the DNA loopdomain containing the D4Z4 repeat cluster in human primary myo-blasts and in murine–human hybrids. A nuclear matrix attachmentsite was found located in the vicinity of the repeat. Prominent innormal human myoblasts and nonmuscular human cells, this site ismuch weaker in muscle cells derived from FSHD patients, suggestingthat the D4Z4 repeat array and upstream genes reside in two loopsin nonmuscular cells and normal human myoblasts but in only oneloop in FSHD myoblasts. We propose a model whereby the nuclearscaffold�matrix attached region regulates chromatin accessibility andexpression of genes implicated in the genesis of FSHD.

D4Z4 � nuclear matrix � heterochromatin � transcription

Fascioscapulohumeral muscular dystrophy (FSHD) is an auto-somal dominant disease with a prevalence of 1:20,000 (1).

FSHD is characterized by weakness and atrophy of muscles of theface, upper arms, and shoulder girdle. Linkage analysis has mappedthe FSHD locus to the subtelomeric region of the long arm ofchromosome 4 (2).

The disorder is related to a short repeat array that remains afterdeletion of an integral number of tandemly arrayed 3.3-kb repeatunits on chromosome 4. The size of this polymorphic locus (D4Z4)varies in normal individuals from 35 to 300 kb, whereas in FSHDpatients it is consistently shorter than 35 kb (3). Partial deletion ofthe D4Z4 array on chromosome 4 ultimately leads to FSHD and iscurrently used as a diagnostic tool in genetic counseling to predictthe probability of the disease (1, 3–5). A correlation exists betweenthe extent of the deletion and its clinical expression: Indeed,patients with one to three repeats develop an early FSHD, whereasindividuals with nine to 10 repeats exhibit a weaker form of thedisease (5).

Extensive efforts to identify gene transcripts associated with the4q35-specific D4Z4 repeat, as potential FSHD candidate genes,have been largely unsuccessful (6). The 3.3-kb D4Z4 elementscontain a cryptic DUX4 gene potentially coding for a doublehomeodomain protein (7), and an overall perturbation of mRNAexpression profiles can be observed in FSHD patients (8–10), butthe disease appears to result from an as yet unexplained mechanismwith a genetic alteration not residing within a causative gene for thedisease.

The 4q35 genomic region (Fig. 1) displays heterochromaticfeatures and might exert repressive effects on neighboring geneswith a mechanism similar to position effect variegation. A de-creased D4Z4 repeat number consistently results in inappropriateup-regulation of adjacent FRG2, FRG1, and Ant1 in FSHD muscle(11–13). It has also been shown that a transcriptional repressorcomplex binds D4Z4, whose deletion would trigger overexpressionby lack of repression (11). Indeed, overexpression of FRG1 in

transgenic mice provokes a phenotype similar to that of FSHD (14).However, such a model of position effect has been recently chal-lenged in two reports of an apparent lack of up-regulation of any4q35 gene and because of the histone H4 acetylation state in FSHDlymphoid cells (9, 10). The region is also hypomethylated in FSHDpatients myoblasts as compared with normal tissues, suggesting thatthe chromatin status may be different (15).

According to a recent study of histone H4 acetylation, D4Z4exhibits properties of unexpressed euchromatin (16). Another studyhas not revealed any significant differences in chromatin organi-zation at 4q35.2 between normal and FSHD myoblasts. It is difficultto draw conclusions from these data, however, because they relateto regions 300 and 850 kb away from the D4Z4 array on chromo-some 4 (17). Recently, distal regions of 4q35 have been shown to beassociated with peripheral heterochromatin (18). Earlier studiesalso had demonstrated an association of D4Z4 with heterochro-matin (19). Extended studies of DNA domains show the existenceof short blocks of heterochromatin that are easy to overlook bypartial analysis but that nevertheless play an important role in generegulation (20). Therefore, it appeared important to compare thelarge-scale chromatin organization of the 4q35 locus between cellsfrom FSHD patients and normal human myoblasts.

In eukaryotic nuclei and metaphase chromosomes, DNA isorganized into loop domains (21). These loops are anchored to thenuclear skeleton or matrix via specific sequences, called nuclearscaffold�matrix attached regions (S�MARs) (22, 23). S�MARs,generally A�T-rich, are located within fragments ranging from 200to 1,000 bp. Some S�MARs reside in nontranscribed regions,sometimes within introns, whereas others are function-related andfound in the vicinity of enhancers, insulators, replication origins,and transcribed genes (see ref. 24 for a review). We have shownearlier that association of S�MARs with the nucleoskeleton maychange during development (25).

To evaluate the possible effect of chromatin organization ofD4Z4 on genes located at a distance to the D4Z4 repeat array, wehave studied the large-scale organization of the 4q35 locus. We havefound that it contains an upstream S�MAR and forms an inde-pendent loop in nonmuscular cells and in normal myoblasts. Incontrast, in myoblasts from FSHD patients, the D4Z4 array and theupstream genes form a single loop domain, thus allowing forcis-regulation of these genes.

ResultsA Strong S�MAR Is Located Upstream of the D4Z4 Repeat in the 4q35Locus. Long-range chromatin organization plays an important rolein the organization of the genome for replication and transcription

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: FSHD, facioscapulohumeral muscular dystrophy; S�MAR, nuclear scaffold�matrix attached region; FRR MAR, FSHD-related region S�MAR.

‡To whom correspondence should be addressed. E-mail: [email protected].

© 2006 by The National Academy of Sciences of the USA

6982–6987 � PNAS � May 2, 2006 � vol. 103 � no. 18 www.pnas.org�cgi�doi�10.1073�pnas.0511235103

(24). Chromatin loop domains represent one of the highest levels ofDNA organization within the genome. The loops are delimited bythe S�MARs.

We have studied the organization of the D4Z4 array andsurrounding sequences by analyzing the nuclear matrix attachmentin the region. We prepared the fraction of nuclear matrix-associatedDNA by DNase I treatment of the isolated nuclei followed byextraction of soluble proteins with 2 M NaCl. The resulting nuclearmatrix contains the sites of DNA attachment to the nuclear matrix(for details, see ref. 26). The DNA associated with the nuclearmatrix was purified, radiolabeled, and used as a probe to examinethe organization of the nuclear matrix attachment sites within theD4Z4 loop domain.

A recombinant plasmid pGEM42 containing two D4Z4 repeatsand 5� and 3� flanking sequences was digested with HincII and KpnIrestriction endonucleases (Fig. 2A). The digested DNA was thenseparated on agarose gels (Fig. 2B). The gels were blotted andprobed with either total human DNA (Fig. 2C) or nuclear matrix-associated DNA from HeLa cells (Fig. 2D).

HeLa matrix-associated DNA strongly hybridizes to two D4Z4

locus restriction fragments, corresponding to the flanking sequenceand the D4Z4 array (Fig. 2D), whereas total human DNA stronglyhybridizes with D4Z4 repeats and the repetitive sequence pLAM(Fig. 2C) and weakly hybridizes to the unique sequences (data notshown). Therefore, (i) the D4Z4 repeats are associated with thenuclear matrix, and (ii) they are flanked by an S�MAR on thecentromeric side of the repeat array, thus physically separatingthe D4Z4 repeats from upstream genes. We designated the 400-bpS�MAR located in the proximal region of the D4Z4 repeat asFSHD-related region S�MAR (FRR MAR). A sequence flankingthe D4Z4 array on the telomeric side, pLAM, is also weaklyassociated with the nuclear matrix.

The human genome contains sequences homologous to D4Z4and pLAM on several chromosomes in addition to chromosome 4(4, 19, 27), and FRR MAR is present in two copies in the genome:on chromosome 4 and in the homologous region on chromosome10. It was therefore possible that the interaction detected betweenthe nuclear matrix and the D4Z4 array in our assay could have takenplace not at 4q35 but rather at one or more of these otherchromosomal loci. To address this possibility, we used a human�rodent hybrid cell line, GM10115A, containing a single humanchromosome 4. Indeed, the S�MARs are highly conserved betweenspecies (28, 29); therefore, one can study chromatin organization insuch hybrids. The rodent genome lacks D4Z4 repeats (30); thus, theonly genomic copy of D4Z4 and adjacent sequences originates from4q35 of the human chromosome 4. Fig. 2E shows that, in thenuclear matrix preparations from GM10115A cells, D4Z4 is weaklyassociated with the nuclear matrix. The association of the distalS�MAR corresponding to the pLAM repeat was somewhat stron-ger in GM10115A cells than in HeLa cells. The attachment of theubiquitous and non-species-specific S�MAR from the upstreamregion of the c-myc gene (29, 31) to the nuclear matrix was used tonormalize the exposures. On the basis of these results, we concludethat the FRR MAR derived from chromosome 4 is indeed asso-ciated with the nuclear matrix and flanks the D4Z4 repeat arraywithin the 4q35 locus.

FRR MAR Association with the Nuclear Matrix Is Strongly Diminishedin Primary Myoblasts from FSHD Patients. We have tested forpotential changes in the association of the 4q35 locus to the nuclearmatrix between cultured primary myoblasts from FSHD patientsand from normal myoblasts. A significant decrease in the associa-tion of the FRR MAR with the nuclear matrix was observed inpatients’ myoblasts as compared with normal ones (Fig. 2 F and G).Interaction of pLAM with the nuclear matrix was diminished bothin the normal primary human myoblasts and in myoblasts fromFSHD patients, as compared with non-muscular HeLa andGM10115A cells, whereas association of the D4Z4 repeat was alsomuch weaker than in HeLa cells. A similar pattern was observed innormal differentiated myoblasts and in differentiated myoblastsfrom FSHD patients (data not shown). The association of the FRRMAR was diminished �30 � 6% in myoblasts from FSHD patients,suggesting that one FRR MAR of four on either chromosomes 4or 10 is dissociated from the nuclear matrix.

Fig. 1. Organization of the 4q35 locus.

Fig. 2. Association of the 4q35 locus with the nuclear matrix in normal cells andin primary myoblasts derived from FSHD patients. (A) Map of recombinant DNAused for hybridization with nuclear matrix DNA (E, EcoRI; K, KpnI; H, HincII). (B)Electrophoretic pattern of restriction fragments. Numbers indicate the positionsof the restriction fragments on the map in A. (C) Hybridization with total DNA.Theblot isunderexposedtorevealDNArepeats. (D–G)Hybridizationwithnuclearmatrix-associated DNA from HeLa cells, nuclear matrix-associated DNA fromGM10115A cells (E), nuclear matrix-associated DNA from normal human myo-blasts (F), and nuclear matrix-associated DNA from myoblasts derived from anFSHD patient (G). Arrows indicate the positions of the human and murine c-mycS�MARs used as controls, the FRR MAR, and the pLAM.

Petrov et al. PNAS � May 2, 2006 � vol. 103 � no. 18 � 6983

GEN

ETIC

S

Taken together, our data indicate that the loop domainorganization at the 4q35 locus in myoblasts of normal subjects isdifferent from that of FSHD patients.

Mapping the Chromatin Loop Domain at the 4q35 Locus by UsingGenomic DNA Array. Next we undertook the mapping of the chro-matin loop organization in the 250-kb region between D4Z4repeats and the upstream area of the FRG1 gene in normalmyoblasts and in cells from FSHD patients by using genomic DNAarrays. We have used a technique for mapping the interactions ofDNA with the nuclear matrix based on oligonucleotide DNA arrays(32). This method allows for rapid and accurate study of S�MARsover large sequenced areas of various genomes.

The array covers 250 kb of the 4q35.2 locus between the D4Z4and the upstream area of the FRG1 gene (Fig. 1). The oligonucle-otides have been numbered according to their distance (in kilo-bases) from the first proximal D4Z4 repeat in the array. Theoligonucleotides were quantified and slot-blotted onto a nylonmembrane as described in ref. 32. As a control, the chosenoligonucleotides in the array were tested for the presence ofrepetitive DNA by hybridization with total DNA. Total humanDNA hybridizes almost equally to the 4q35 locus array (Fig. 3A).We concluded that the chosen oligonucleotides do not containmultiple-copy DNA repeats.

Chromosome 4 contains large regions of homology with chro-mosome 10 within the 4q35 locus. To double-check the array, wehybridized it with the total DNA extracted from the GM10115Ahybrid cell line containing human chromosome 4. The pattern ofhybridization was largely similar to that of total human DNA, thusvalidating the specificity of our array (Fig. 3B). Somewhat strongerhybridization of the murine–human hybrid cell line with theoligonucleotides at 163 and 151 bp can be explained by thehomologies of the oligonucleotides and the murine DNA.

In contrast, the nuclear matrix-associated DNA from the normalmyoblasts shows a specific hybridization pattern: The association ofthe 4q35 locus with the nuclear matrix becomes extremely specific,restricted to two strong sites located at 4 and 165 bp relative to theD4Z4 array (Fig. 3C). The attachment at 4 bp corresponds to theFRR MAR, in perfect agreement with our previous experiments.Consistently, a decrease was observed in the attachment of thenuclear matrices from FSHD patients at 4 bp. Interestingly, thedistal border of the DNA loop was also different and located at 171

kb in FSHD patients, compared with 165 kb in normal humanmyoblasts.

Hybridization of the soluble DNA fraction with the array re-vealed a pattern similar to that obtained with total DNA (data notshown). This similarity is not surprising given that the non-matrix-associated DNA constitutes 95–98% of the genome.

We conclude that there exists a specific organization of the 4q35locus in human myoblasts with the loop domain ends located at171–165 and 4 kb relative to the D4Z4 array. Therefore, the D4Z4repeats and neighboring genes (FRG2 and FRG1) are located intwo distinct loop domains and are physically separated by the FRRMAR. Dissociation of the FRR MAR on the defective chromo-some 4 in myoblasts from the FSHD patients from the nuclearmatrix may change the overall configuration of the loop domain,bringing together the D4Z4 array and the neighboring genes. Thisevidence conclusively identifies a changed chromatin organizationbetween the cells of FSHD patients and the nonaffected cells.

D4Z4 and Upstream Genes Within the 4q35 Locus Are Located in TwoDistinct Loops in Non-Myoblast Cells and on ‘‘Normal’’ Chromosome 4in FSHD Patients. The DNA loops fixed at the nuclear matrix orscaffold can be visualized in histone-depleted nuclei as a halo ofDNA surrounding the nuclear matrix (33). Using FISH on high-saltextracted nuclei (nuclear halos) or chromosomes, one can detectindividual loops and determine whether short individual sequencesare located on the nuclear matrix or in the DNA loops (32, 34–38).

The experiments on biochemical mapping of the S�MAR in theproximity of the D4Z4 repeats suggest that D4Z4 and the upstreamgenes are located in two distinct DNA loop domains in normalprimary myoblasts, whereas they comprise a single domain in thedamaged chromosome in FSHD patients.

We have used FISH to further confirm these observations.Nuclear halos were prepared by salt extraction of nuclei immobi-lized on glass slides. The blue halo corresponds to DNA loopsoriginating from the nuclear matrix (Fig. 4). The nuclear matrix canalso be visualized in these preparations by staining with lamins, asignal that coincides with bright DAPI staining (data not shown).

A PCR-amplified DNA fragment corresponding to the FRRMAR was labeled and used as a probe in FISH with the nuclearhalos or matrices from normal myoblasts. Hybridization was vir-tually restricted to the nuclear matrix (Fig. 4A; see also Fig. 7 A andB, which is published as supporting information on the PNAS web

Fig. 3. Mapping the nuclear ma-trix attachment site in the 4q35locus by DNA array technique. (Aand B) Hybridization of the totalhuman DNA (A) and total DNAfrom the mouse– human hybridcell line GM11015A (B) with agenomic DNA array covering 250kb in the 4q35 locus. (C) The re-sults of hybridization of nuclearmatrix-associated DNA from cul-tured primary myoblasts fromFSHD patients (white columns)and normal subjects (black col-umns). The hybridization datawere normalized against a posi-tive control. Hybridization to aS�MAR from the human c-mycgene locus in A and C and againstmurine c-myc S�MAR in B was as-signed a value of 1. The average ofthree independent experiments(two hybridizations per experi-ment) is presented.

6984 � www.pnas.org�cgi�doi�10.1073�pnas.0511235103 Petrov et al.

site). In 57 cells inspected, 89% of the FRR MAR probe wasdetected on the nuclear matrix, thus validating the results ofS�MAR analysis (Table 1, which is published as supporting infor-mation on the PNAS web site).

In contrast, hybridization of the same probe with nuclear halosand matrices isolated from FSHD-derived myoblasts resulted in adifferent distribution of the signal (Figs. 4B and 7 C and D). In 51nuclei inspected, only 67% of signals were located within thenuclear matrix, whereas 33% of signals were found on loop regionof the halos. Similar results have been obtained by using in situdetection of the FRR MAR on the DNaseI-treated nuclear ma-trices: four FRR MAR signals were detected in nuclear matricesfrom normal cells vs. three in FSHD cells (data not shown).

The present FISH analysis shows that, in 89% of the cases, thedelocalized FRR MAR is associated with a short D4Z4 array,suggesting that it is the deleted chromosome 4 that is delocalizedfrom the nuclear matrix. The probe corresponding to D4Z4 gavesignals preferentially within loop DNA in both types of myoblasts,again as one would expect on the basis of the results of biochemicalanalysis. In 51 cells inspected, 88% of the signals given by the probewere detected in loop DNA. The presence of 12% of the signals onthe nuclear matrix area most probably reflects distortion of thethree-dimensional loop halos during immobilization on the micro-scopic slide. Indeed, in human–murine hybrid cell line GM10115Acontaining a single D4Z4 array, the signals derived from the D4Z4

probe in contrast to the FRR MAR probe are present in 98% ofthe nuclear halos.

Together, these observations confirm the delocalization of de-fective chromosome 4 from the nuclear matrix, which may result indrastic changes in both chromatin structure and transcriptionalregulation with a probable role in FSHD.

Visualization of a Chromatin Loop Domain in the 4q35 Locus. We thenattempted to visualize individual loops containing the D4Z4 up-stream genes by using as a probe a bacterial artificial chromosomeclone with an insert of 160 kb covering the 4q35 locus betweenpositions 191108570 and 191272691 (�90–250 kb relative to theD4Z4 array) (see Fig. 1 for details).

In these conditions, most of the observed loops were V-shaped(Fig. 5; see also Fig. 8, which is published as supporting informationon the PNAS web site). This shape corresponds to two incompleteloops on each side of a S�MAR in the middle, consistent with thefact that the bacterial artificial chromosome clone used did notcover the entire loop, as mapped by the array technique.

Incomplete loops were observed in 73% of inspected halos. In27% of the nuclei, the signals were distributed in a disorderedfashion over both the core and halo (data not shown), which is likelyto result from distortion of the halo during preparation and�or froman unfavorable loop position on the plane surface of the slide. Onlyone S�MAR was observed in this region. No visible difference inloop organization of the distal end of the loop was observedbetween primary myoblasts from healthy subjects and FSHDpatients, thus confirming the results of our biochemical analysis.Indeed, the resolution of the FISH technique is not sufficient todetect a subtle difference (6 kb) between the attachment siteslocated at positions 171 and 165, respectively.

We have roughly estimated the size of incomplete loop branchesin case of both types of studied myoblasts, measured by tracing theircontour (Fig. 5). Each loop of such shape reveals almost equivalentarms, which would correspond to the MAR position at �170 kbrelative to D4Z4 array, whereas we have observed the S�MARspositioned at positions 165–171 by using the genomic DNA arraytechnique.

DiscussionDrastic Changes in Chromatin Loop Domain Organization of the 4q35Locus in Myoblasts from Patients: Implications for the FSHD. Theresults presented here clearly demonstrate that normal humanprimary myoblasts and myoblasts derived from FSHD patients

Fig. 5. Visualization of DNA loops from the 4q35 locus. Shown are the resultsof hybridization of the bacterial artificial chromosome probe that contains aninsert covering the region between 90 and 250 kb relative to the D4Z4 array(green) of the nuclear halos from human primary myoblasts of unaffected indi-viduals (A–A�) or FSHD patients (B–B�). (A and B) DNA stained with DAPI. (A� andB�) The results of hybridization. (A� and B�) Merged images. (A� and B�) The pathsof chromatin loops (green) relative to the nuclear matrices and halos (blue).

Fig. 4. FISH on the nuclear halos from the primary myoblasts from normal subjects and FSHD patients. Shown are the results of hybridization of the FRR MAR(red) and the D4Z4 repeat (green) to the nuclear halos from human primary myoblasts from unaffected individuals (A) and those from FSHD patients (B). Thepositions of the FISH signals corresponding to FRR MAR are indicated by arrows. The first image in each row shows the nuclei and nuclear halos�matricescounterstained with DAPI (blue); the second image shows FISH signals from FRR MAR; the third image shows FISH signals corresponding to D4Z4; the fourth imageshows the merged results of the two previous images; and the fifth image shows paths of chromatin loops (green) relative to the nuclear matrices and halos (blue).

Petrov et al. PNAS � May 2, 2006 � vol. 103 � no. 18 � 6985

GEN

ETIC

S

present with specific organizations of the 4q35 locus into DNA loopdomains and that these organizations differ.

Several models for the molecular basis of FSHD have beenproposed. One model is based on the existence of a repressiveelement (D4Z4-binding element) within the D4Z4 repeat thatbinds a repressor complex (D4Z4-repressing complex), resulting intranscriptional repression spreading onto neighboring sequences.According to this model, deletion of an integral number of D4Z4repeats in FSHD patients would reduce the amount of boundrepressor complexes and consequently decrease (or abolish) tran-scriptional repression of 4q35 genes (11). Thus, the deletion ofrepeated elements in the subtelomeric 4q region would act in cis onneighboring genes, derepressing transcription and starting a cas-cade of events that ultimately leads to FSHD.

According to other studies, no significant difference in chromatinorganization can be detected in the 4q35.2 locus between normalhuman primary myoblasts and myoblasts derived from FSHDpatients. It appears difficult to draw any conclusions from thesedata, because they were focused on regions 300 and 850 kb away,respectively, from the chromosome 4 D4Z4 array (16, 17).

According to another study, the D4Z4 array was found to beassociated with the periphery of the nucleus (18). Unfortunately,these investigations carried out in cells derived from FSHD patientswere done in cells of lymphoid, not muscular, origin, making it lessrelevant for exploring the mechanistic substratum of defects ex-pressed in myoblasts only.

The present data therefore provide evidence of clear-cut differ-ences between the deleted and nondeleted chromosomes 4 inmyoblasts from FSHD patients as well as differences between adeleted chromosome 4 from FSHD patients and the correspondingsubtelomeric region of chromosome 4q in cultured primary myo-blasts derived from normal individuals. The D4Z4 array and itsneighboring genes, namely FRG2 and FRG1, were found to beincluded in two distinct loop domains in control primary myoblastsas well as in the normal chromosome 4 in FSHD patients. Incontrast, in the defective chromosome 4q of FSHD patients, thesegenes were located within the same chromatin loop domain as thepartially deleted D4Z4 repeat array.

We postulate that these differences may account for changesobserved in transcriptional pattern at the 4q35 locus. Indeed, ourprevious data suggest that D4Z4 has the properties of a tran-scriptional enhancer (ref. 39 and A.P. and Y.S.V., unpublisheddata) and might enhance transcription from the FRG2 promoter(13). The presence of the D4Z4 enhancer within the same loopdomain as the FSHD candidate genes FRG2 and FRG1, along withthe reduction in size of the repeat array containing the repressiveD4Z4-binding element, could up-regulate transcription of neigh-boring genes in affected individuals, thereby resulting in FSHD inthese patients (Fig. 6).

It is also noteworthy that a subtle difference exists at the distal

end of the loop domain. In primary myoblasts of normal individuals,the 5� S�MAR is located at position 171 in the array, whereas it liesat position 165 in myoblasts from FSHD patients. This phenome-non, referred to as ‘‘sliding,’’ may be linked to the selection of anS�MAR better adapted to a specific function or transcriptionalpattern, as suggested by Heng et al. (40). Further studies will benecessary to determine whether this phenomenon plays a role in thegenesis of FSHD.

4qA�4qB Genotypes and Long-Range Chromatin Organization. Apolymorphic segment of 10 kb directly distal to D4Z4 was shown toexist in two allelic forms, 4qA and 4qB. Although both alleles areequally common in the general population, it has been reported thatFSHD is associated only with the 4qA allele (41, 42). It would beinteresting to see whether this polymorphism affects the associationwith the nuclear matrix of the region located between the D4Z4array and the telomere. Unfortunately, no coherent sequence dataexist on this region, making it impossible to use the array technique;however, classical biochemical technique may prove useful.

S�MARs and Reduction of the D4Z4 Array by Recombination. The nearidentity of the subtelomeric parts of chromosomes 4q and 10qmakes it likely that the mechanism of partial deletion of the D4Z4array on chromosome 4 occurs through a translocation or recom-bination with chromosome 10 (43–45). The presence of a strongS�MAR near the D4Z4 array may favor such a recombination.

In many recombination-prone regions, including the MLL geneand the IFN-II loci, the recombination hot spots are located in thevicinity of S�MARS (for reviews, see refs. 46 and 47). Recombi-nation enhanced by adjacent matrix attachment regions may resultin the loss of D4Z4 repeats. This hypothesis clearly calls for furtherinvestigation.

Relationship Between Heterochromatin and Matrix Attachment. Thequestion of a relationship between matrix attachment and hetero-chromatin has been widely discussed (for a review, see ref. 48). Itis generally believed that S�MARs may stop the expansion ofheterochromatin and protect neighbor genes that are being tran-scribed from heterochromatinization. Indeed, inserting a S�MARinto a construct carrying a (reporter) gene generally results inenhanced transcription in transgenic animals or plants, in relationwith changes observed in the chromatin structure proximal anddistal to the integrated gene (49). The assembly of heterochromatinat S�MARs has also been implicated in the function of theimprinting center at human chromosome 15q11–15q13 (50). Con-trasting with these views, our results suggest that S�MARs do notconstitute efficient barriers for heterochromatinization. Indeed,heterochromatin was found to spread over the FRR MAR innormal human myoblasts (19). In FSHD patients, in whom theheterochromatin structure seems to be altered under the effect ofa reduced D4Z4 array (11), the FRR MAR appears not to beassociated with the nuclear matrix.

The changes in long-range chromatin organization betweennormal myoblasts and those from FSHD patients reported in thepresent study may reveal perspectives in the study of the disease.

Materials and MethodsCells. HeLa cell line was purchased from American Type CultureCollection. GM10115A hybrid murine cell line containing humanchromosome 4 was the kind gift of R. Tupler (Universita delgi Studidi Modena e Reggio Emilia, Modena, Italy).

Primary myoblasts from two healthy individuals and two FSHDpatients with 5.5 D4Z4 repeats and seven repeats in the 4q35 arraywere cultured on collagen-coated support in DMEM supplementedwith 20% bovine fetal serum.

Nuclei and Nuclear Matrices. Nuclei from primary human myoblastsand HeLa and GM10115A cells were isolated as described in ref.

Fig. 6. Loop domain organization and transcriptional control in the 4q35 locus.

6986 � www.pnas.org�cgi�doi�10.1073�pnas.0511235103 Petrov et al.

26. Nuclear matrices were prepared by treatment of the isolatednuclei with NaCl as follows: digestion buffer (100 mM NaCl�25 mMKCl�10 mM Tris�HCl, pH 7.5�0.25 mM spermidine) was added to105 nuclei to a final volume of 400 �l. DNase I was added to a finalconcentration of 100 �g/ml, and the samples were digested for 2 hat 4°C, followed by the addition of CuCl2 to a final concentrationof 1 mM for 10 min at 4°C. The nuclei were then extracted byaddition of one volume of an EB buffer (4 M NaCl�20 mMEDTA�40 mM Tris�HCl, pH 7.5). The resulting nuclear matriceswere washed (2 M NaCl�10 mM EDTA�20 mM Tris�HCl, pH 7.5),and nuclear matrix-associated DNA was extracted after proteinaseK treatment and either radioactively labeled by using the Ready-to-Go kit (AP Biotech) or labeled with DIG by using a DIG-High Prime kit (Roche Diagnostics) and used as a probe forhybridization.

DNA Array. The DNA array consisted of 102 35- to 45-mer oligo-nucleotides spaced �2 kb apart spanning the region from D4Z4repeat array to the far upstream region of the FRG1 gene. The listof oligonucleotides is available upon request. The distal part of thearray surrounding the FRG1 gene contained the unique chromo-some 4-specific sequences. The proximal part contained sequencesspecific for chromosomes 4 and 10 due to 99% homology betweenthese regions. The oligonucleotides were slot-blotted onto HybondN� filters (Amersham Pharmacia) and hybridized at 40.5°C over-night. The blot was incubated with the anti-DIG antibodies (Roche)and revealed by using an enhanced chemiluminescence kit (ECL�;Amersham Pharmacia). The films were scanned and quantifiedwith IMAGE GAUGE 4.0 (Fuji). The hybridization data were normal-ized against S�MAR from the human or murine c-myc gene loci.The experiments were carried out in triplicate. Data from twoindependent experiments are presented.

Nuclear Halo�Matrices Preparation for FISH. Nuclei were prepared asdescribed in ref. 26. To obtain the nuclear halos, nuclei werepelleted at 200 � g for 10 min onto glass slides. Slides were then

incubated in buffer H1 (10 mM Pipes, pH 6.8�0.1 M NaCl�0.3 Msucrose�3 mM MgCl2�0.5% Triton X-100�0.1 mM CuSO4�1 mMPMSF) for 10 min on ice, followed by treatment in buffer H2 (1 mMPipes, pH 6.8�2 M NaCl�10 mM EDTA�0.1% digitonin�0.05 mMspermine�0.125 mM spermidine) for 4 min. Slides were then passedthrough 10�, 5�, 2�, and 1� PBS followed by 10%, 30%, 50%,70%, and 95% ethanol solutions, air-dried, and finally fixed at 70°Cfor 2 h.

Preparation of nuclear matrices on the microscope slides wascarried out essentially as described in ref. 51.

FISH Analysis. FRR MAR was amplified by PCR with the pGEM42plasmid and labeled with DIG-11-dUTP (Roche Diagnostics). TheD4Z4 probe was derived from pGEM42 (51) and labeled withbiotin-14 as well as BAC RP11521G19 located 100 kb proximal toD4Z4 array on 4q35.

Hybridization to slides was performed as described in ref. 52 byusing anti-biotin mouse antibodies conjugated with Alexa Fluor 488(Molecular Probes) or anti-DIG sheep antibodies conjugated withTAMRA (Roche). The nuclei, nuclear halos, and matrices werecounterstained by DAPI in VECTASHIELD mounting medium(Vector Laboratories). Slides were examined on an Olympus Provisfluorescence microscope with a 60� oil immersion objective andappropriate filters. Images were captured with a charge-coupleddevice camera (Photometrics, Tucson, AZ), using RSIMAGE soft-ware (Scanalytics, Billerica, MA).

We thank Dr. A. Belayev (Universite de Mons-Hainaut, Mans, Belgium)for the kind gift of the pGEM42 plasmid, Dr. R. Tupler for the kind giftof the GM10115A strain, and Drs. A. Hair and V. Ogryzko for criticalreadings of the manuscript. This work was supported by grants from theAssociation Francaise contre les Myopathies (to Y.S.V.). A.P. wassupported by postdoctoral fellowships from The Federation of Biochem-ical Societies and the Fondation pour la Recherche Medicale. I.P. wassupported by a postdoctoral fellowship from the Association Francaisecontre les Myopathies. D.L. was suppored by the Association Francaisecontre les Myopathies.

1. Lunt, P. W. & Harper, P. S. (1991) J. Med. Genet. 28, 655–664.2. Wijmenga, C., Padberg, G. W., Moerer, P., Wiegant, J., Liem, L., Brouwer, O. F., Milner,

E. C., Weber, J. L., van Ommen, G. B., Sandkuyl, L. A., et al. (1991) Genomics 9, 570–575.3. van Deutekom, J. C., Wijmenga, C., van Tienhoven, E. A., Gruter, A. M., Hewitt, J. E.,

Padberg, G. W., van Ommen, G. J., Hofker, M. H. & Frants, R. R. (1993) Hum. Mol. Genet.2, 2037–2042.

4. Wijmenga, C., Hewitt, J. E., Sandkuijl, L. A., Clark, L. N., Wright, T. J., Dauwerse, H. G.,Gruter, A. M., Hofker, M. H., Moerer, P., Williamson, R., et al. (1992) Nat. Genet. 2, 26–30.

5. Tawil, R. & van der Maarel, S. (February 6, 2006) Muscle Nerve, 10.1002/mus.205226. Hewitt, J. E., Lyle, R., Clark, L. N., Valleley, E. M., Wright, T. J., Wijmenga, C., van Deutekom,

J. C., Francis, F., Sharpe, P. T., Hofker, M., et al. (1994) Hum. Mol. Genet. 3, 1287–1295.7. van Geel, M., Heather, L. J., Lyle, R., Hewitt, J. E., Frants, R. R. & de Jong, P. J. (1999)

Genomics 61, 55–65.8. Tupler, R., Perini, G., Pellegrino, M. A. & Green, M. R. (1999) Proc. Natl. Acad. Sci. USA

96, 12650–12654.9. Winokur, S. T., Chen, Y. W., Masny, P. S., Martin, J. H., Ehmsen, J. T., Tapscott, S. J., Van

Der Maarel, S. M., Hayashi, Y. & Flanigan, K. M. (2003) Hum. Mol. Genet. 12, 2895–2907.10. Wohlgemuth, M., Lemmers, R. J., Van Der Kooi, E. L., Van Der Wielen, M. J., Van

Overveld, P. G., Dauwerse, H., Bakker, E., Frants, R. R., Padberg, G. W. & Van Der Maarel,S. M. (2003) Neurology 61, 909–913.

11. Gabellini, D., Green, M. R. & Tupler, R. (2002) Cell 110, 339–348.12. Laoudj-Chenivesse, D., Carnac, G., Bisbal, C., Hugon, G., Bouillot, S., Desnuelle, C.,

Vassetzky, Y. & Fernandez, A. (2005) J. Mol. Med. 83, 216–224.13. Rijkers, T., Deidda, G., van Koningsbruggen, S., van Geel, M., Lemmers, R. J., van

Deutekom, J. C., Figlewicz, D., Hewitt, J. E., Padberg, G. W., Frants, R. R. & van derMaarel, S. M. (2004) J. Med. Genet. 41, 826–836.

14. Gabellini, D., D’Antona, G., Moggio, M., Prelle, A., Zecca, C., Adami, R., Angeletti, B., Ciscato,P., Pellegrino, M. A., Bottinelli, R., Green, M. R. & Tupler, R. (2005) Nature 439, 973–977.

15. van Overveld, P. G., Lemmers, R. J., Sandkuijl, L. A., Enthoven, L., Winokur, S. T., Bakels,F., Padberg, G. W., van Ommen, G. J., Frants, R. R. & van der Maarel, S. M. (2003) Nat.Genet. 35, 315–317.

16. Jiang, G., Yang, F., van Overveld, P. G., Vedanarayanan, V., van der Maarel, S. & Ehrlich,M. (2003) Hum. Mol. Genet. 12, 2909–2921.

17. Yang, F., Shao, C., Vedanarayanan, V. & Ehrlich, M. (2004) Chromosoma 112, 350–359.18. Tam, R., Smith, K. P. & Lawrence, J. B. (2004) J. Cell Biol. 167, 269–279.19. Winokur, S. T., Bengtsson, U., Feddersen, J., Mathews, K. D., Weiffenbach, B., Bailey, H.,

Markovich, R. P., Murray, J. C., Wasmuth, J. J., Altherr, M. R., et al. (1994) ChromosomeRes. 2, 225–234.

20. Prioleau, M. N., Nony, P., Simpson, M. & Felsenfeld, G. (1999) EMBO J. 18, 4035–4048.21. Buongiorno-Nardelli, M., Micheli, G., Carri, M. T. & Marilley, M. (1982) Nature 298, 100–102.22. Cockerill, P. N. & Garrard, W. T. (1986) Cell 44, 273–282.23. Mirkovitch, J., Mirault, M. E. & Laemmli, U. K. (1984) Cell 39, 223–232.24. Vassetzky, Y., Lemaitre, J. M. & Mechali, M. (2000) Crit. Rev. Eukaryotic Gene Expression 10, 31–38.

25. Vassetzky, Y., Hair, A. & Mechali, M. (2000) Genes Dev. 14, 1541–1552.26. Gasser, S. M. & Vassetzky, Y. S. (1998) in Chromatin: A Practical Approach, ed. Gould, H.

(Oxford Univ. Press, Oxford), pp. 111–124.27. Lyle, R., Wright, T. J., Clark, L. N. & Hewitt, J. E. (1995) Genomics 28, 389–397.28. Cockerill, P. N. & Garrard, W. T. (1986) FEBS Lett. 204, 5–7.29. Girard-Reydet, C., Gregoire, D., Vassetzky, Y. & Mechali, M. (2004) Gene 332, 129–138.30. Clark, L. N., Koehler, U., Ward, D. C., Wienberg, J. & Hewitt, J. E. (1996) Chromosoma

105, 180–189.31. Gromova, I. I., Thomsen, B. & Razin, S. V. (1995) Proc. Natl. Acad. Sci. USA 92, 102–106.32. Ioudinkova, E., Petrov, A., Razin, S. V. & Vassetzky, Y. S. (2005) Genomics 85, 143–151.33. Vogelstein, B., Pardoll, D. M. & Coffey, D. S. (1980) Cell 22, 79–85.34. Bickmore, W. (1996) Exp. Cell Res. 229, 198–200.35. Byrd, K. & Corces, G. (2003) J. Cell Biol. 162, 565–574.36. Iarovaia, O. V., Bystritskiy, A., Ravcheev, D., Hancock, R. & Razin, S. V. (2004) Nucleic

Acids Res. 32, 2079–2086.37. Kikyo, N., Wade, P. A., Guschin, D., Ge, H. & Wolffe, A. P. (2000) Science 289, 2360–2363.38. Ratsch, A., Joos, S., Kioschis, P. & Lichter, P. (2002) Exp. Cell Res. 273, 12–20.39. Petrov, A. P., Laoudj, D. & Vassetzky, Y. S. (2003) Genetics 39, 147–151.40. Heng, H. H., Goetze, S., Ye, C. J., Liu, G., Stevens, J. B., Bremer, S. W., Wykes, S. M., Bode,

J. & Krawetz, S. A. (2004) J. Cell Sci. 117, 999–1008.41. Lemmers, R. J., de Kievit, P., Sandkuijl, L., Padberg, G. W., van Ommen, G. J., Frants, R. R.

& van der Maarel, S. M. (2002) Nat. Genet. 32, 235–236.42. Lemmers, R. J., Wohlgemuth, M., Frants, R. R., Padberg, G. W., Morava, E. & van der

Maarel, S. M. (2004) Am. J. Hum. Genet. 75, 1124–1130.43. van Overveld, P. G., Lemmers, R. J., Deidda, G., Sandkuijl, L., Padberg, G. W., Frants, R. R.

& van der Maarel, S. M. (2000) Hum. Mol. Genet. 9, 2879–2884.44. van Geel, M., Dickson, M. C., Beck, A. F., Bolland, D. J., Frants, R. R., van der Maarel, S. M.,

de Jong, P. J. & Hewitt, J. E. (2002) Genomics 79, 210–217.45. Lemmers, R. J., van der Maarel, S. M., van Deutekom, J. C., van der Wielen, M. J., Deidda,

G., Dauwerse, H. G., Hewitt, J., Hofker, M., Bakker, E., Padberg, G. W. & Frants, R. R.(1998) Hum. Mol. Genet. 7, 1207–1214.

46. Bode, J., Benham, C., Ernst, E., Knopp, A., Marschalek, R., Strick, R. & Strissel, P. (2000)J. Cell. Biochem. 35, Suppl., 3–22.

47. Bystritskiy, A. A. & Razin, S. V. (2004) Crit. Rev. Eukaryotic Gene Expression 14, 65–77.48. Podgornaya, O. I., Voronin, A. P., Enukashvily, N. I., Matveev, I. V. & Lobov, I. B. (2003)

Int. Rev. Cytol. 224, 227–296.49. Allen, G. C., Spiker, S. & Thompson, W. F. (2000) Plant Mol. Biol. 43, 361–376.50. Greally, J. M., Gray, T. A., Gabriel, J. M., Song, L., Zemel, S. & Nicholls, R. D. (1999) Proc.

Natl. Acad. Sci. USA 96, 14430–14435.51. Gabriels, J., Beckers, M. C., Ding, H., De Vriese, A., Plaisance, S., van der Maarel, S. M.,

Padberg, G. W., Frants, R. R., Hewitt, J. E., Collen, D. & Belayew, A. (1999) Gene 236, 25–32.52. Cai, S. & Kohwi-Shigematsu, T. (1999) Methods 19, 394–402.

Petrov et al. PNAS � May 2, 2006 � vol. 103 � no. 18 � 6987

GEN

ETIC

S